DNA double-strand breaks (DSB) can arise by ionizing radiation, alkylation damage, and replication, including improper processing of lagging strand intermediates. DNA breaks can be a powerful source of chromosome instability as well as programmed genetic modification. Cells have elaborate systems for dealing with DSBs, including DNA repair and checkpoint arrest to increase the opportunity for repair. DSBs in chromosomes lead to a checkpoint arrest at the G2/M boundary in yeast, which provides further opportunities for repair. DSBs are repaired through homologous recombination, end-joining , and by single-strand annealing at homologous regions beyond the breaks. Nearly all organisms exhibit these repair processes as well as checkpoint arrests. Defects in these processes are often associated with disease in humans.[unreadable] DNA ends must be processed to allow homologous interactions for recombination and single strand annealing. Endjoining involves only local nuclease degradation that enables interaction at microhomologies of only a few bases, The Ku and RAD50/MRE11/XRS2 (R/M/X) complexes of proteins are required for endjoining. In addition, the R/M/X complex functions in the nuclease processing of ends to provide recombination substrates. The R/M/X complex has been proposed to have a structural role that holds broken chromosomes and sister chromatids together through a Rad50/Mre11 hook-bridge structure. The Ku complex, which associates at the ends of breaks prevents excessive processing of broken ends. The balance of Ku and R/M/X can determine the extent and timing of end-processing of DSBs that in turn determines the timing of G2/M arrest adapation to a DSB. We have investigated the consequences of DSBs in various mutants and the mechanisms of handling DSBs. Our approaches have been extended to consider repair in the context of chromosomes. While DNA is the central component of chromosomes, the relationship between DNA and chromosome breaks has not been addressed nor the dynamics of DSB repair in different chromosomes.[unreadable] [unreadable] DSBs IN REAL TIME -- In spite of many genetic and biochemical assays for checkpoint arrest and repair, little is known about the behavior of damaged chromosomes in the arrested cells. Furthermore, the question of the relationship between a DSB in DNA and a cytologically detectable chromosome break, as well as possible genetic controls have never been addressed. We developed a system based in the yeast Saccharomyces cerevisiae that provides for chromosome analysis in real time following the induction of a single DSB by an I-SceI endonuclease under the tight control of a GAL1promoter. We utilized tetR-CFP and LacI-GFP to mark each side of a DSB and Spc29-RFP fusion to identify the spindle poles. These proteins bind multiple repeats of their operater target sequences. This allows us to investigate the development of a chromosome break following DNA scission and the relation to spindle pole separation and sister chromatid separation in wild type and various Ku and R/M/X mutants. We have established that the transition from DNA double-strand break measured at the molecular level to cytologically detectable chromosome break is prevented by the physical tethering function of the R/M/X complex and that the appearance of a chromosome break in vivo requires force that is transmitted through microtubules. We also established that the hook function of RAD50 molecules is required for holding chromosomes together. We found that while the induction of a DSB was nearly 100% efficient, at most only 10 to 15% of the cut chromosomes exhibited a chromosome break. We are now investigating additional components that may be responsible for holding chromosomes together. Surprisingly, there is a cold-sensitive component in a rad50 mutant background that results in an increase to 40 to 50% of the cells having a chromosome break. [unreadable] [unreadable] DIFFERENTIAL DSB REPAIR IN CHROMOSOMES: CHROMOSOMAL VS DNA REPAIR. Chromosomal vs DNA repair. The 16 chromosomes of yeast vary in size with a range between ~250-2000 kb. Since the individual chromosomes can be displayed using pulse-field gel electrophoresis, we reasoned that it would be possible to address repair in individual chromosomes and genetic controls. We established that ethidium bromide staining could be used to assess the amount of DNA in each chromosome. This was followed by direct Southern Analysis of breakage and repair in individual chromosomes. Over the range of sizes from 230 to 1500 MB, induction of DSBs by ionizing radiation appears random which in logarithmically growing cells arrested in G2 was approximately 0.6 breaks/100 Gy/Mb. By comparing the reconstitution of full length chromosomes, and the patterns of breaks following exposure to various doses we are now in a position to evaluate the extent to which repair of breaks is random. The Southern analysis enabled us to address restitution of full-size chromosomes. We found that nearly 90% of the chromosomes that experienced one or a few breaks could be repaired in 1hour for the small chromosomes. For the large, where many more breaks were expected the time for repair was considereably enhanced. The repair requires the RAD50, RAD51 and RAD52 genes. [unreadable] [unreadable] Repair and genetic controls was very different for Chromosome XII. The Chromosome XII-specific difference in repair is likely related to nearly half of this chromosome being composed of ribosomal DNA repeats. We propose that DSBs in these repeats can be repaired by a Rad51 independent, single-strand annealing mechanism rather than exchange mechanisms required for repair between sister chromatids. These results suggest that many of the components necessary for DSB repair are present including the capability for resection.[unreadable] [unreadable] We have examined individual colonies for chromosome aberrations using CGH (Comparative Genome Hybridization). We have found that nearly 30-50% of survivors irradiated in G-2 have an aberration. Through detailed analysis of individual chromosome events we have established that nearly all the breakpoints involve small repeats, either 5kb or 300 bp. Thus, while repair between sister chromatids is highly efficient, these findings demonstrate that repeats in the genome can compete effectively for a DSB containing region. In addition, we have found using CGH that ionizing radiation induced whole chromosomal aneuploidy in about 30% of the survivors.